Eef1a2 for use in the prognosis, diagnosis, and treatment of cancer

ABSTRACT

Methods and kits for diagnosing and prognosticating cancer via detection of EEF1A2 and/or EEF1A2 are provided. Also provided are methods for treating cancer via inhibition of expression and/or activity of EEF1A2 and screening assays to identify new anticancer agents based upon their ability to inhibit EEF1A2 expression and/or activity. The methods and kits of the present invention are particularly useful in the prognosis, diagnosis and treatment of ovarian, breast and colorectal cancer.

This application claims the benefit of priority from U.S. provisionalapplication Ser. No. 60/387,231, filed Jun. 7, 2002, the teachings ofwhich herein are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention provides methods and kits for diagnosing andprognosticating cancer via detection of EEF1A2 and/or EEF1A2. Thepresent invention also provides methods for treating cancer viainhibition of expression and/or activity of EEF1A2, screening assays toidentify new anticancer agents based upon their ability to inhibitEEF1A2 expression and/or activity, and compositions comprising aninhibitor of EEF1A2 expression and/or activity for use in the treatmentof various cancers. The methods, kits and compositions of the presentinvention are particularly useful in the prognosis, diagnosis andtreatment of ovarian and breast cancer, as well as colorectal cancer.

BACKGROUND OF THE INVENTION

In the year 2001, 25,000 North American women were expected to bediagnosed with ovarian cancer. Over half of the women diagnosed withovarian cancer are likely to die of this disease.

Amplifications of 20q13 have been identified in both breast and ovariantumors and have been correlated with poor clinical prognosis andincreased tumor aggressiveness in breast and ovarian cancer (Tanner etal. Clin. Cancer Res. 1995 1:1455-1461; Isola et al. Am. J. Pathol. 1995147:905-911; Courjal et al. Br. J. Cancer 1996 74:1984-1989). Thepresence of four or more copies of 20q13 has been associated with adecreased five-year survival after diagnosis in women with ovariancancer (Diebold et al. J. Pathol. 2000 190:564-571). Specifically, a20-30% fraction of ovarian tumors (Courjal et al. Br. J. Cancer 199674:1984-1989; Sonoda et al. Genes Chromosomes Cancer 1997 20:320-328;and Diebold et al. J. Pathol. 2000 190:564-571) and a 20-40% fraction ofbreast tumors (Kallioniemi et al. Proc. Natl. Acad. Sci. USA 199491:2156-2160; Tanner et al. Cancer Res. 1994 54:4257-4260; and Tanner etal. Cancer Res. 1996 56:3441-3445) have an increase in copy number ofthe 20q13 locus, thus implicating one or more genes at 20q13 in thegenesis and progression of ovarian tumors.

Multiple genes map to the 20q13 locus including, but not limited toZNF217, NABC1 (Collins et al. Proc. Natl. Acad. Sci USA 199895:8703-8708), CYP24 (Albertson et al. Nat. Genet. 2000 25:144-146),STK15/BTAK (Bischoff et al. EMBO J. 1998 17:3052-3065) and aurora2kinase (Bischoff et al. EMBO J. 1998 17:3052-3065). Neither CYP24 norNABC1 are known to have tumorigenic properties. However, mapping of thebreast 20q13 amplicon by CGH (comparative genomic hybridization)suggests that the DNA amplifications center on a 2 Mb region around20q13.2 and CYP24 (Albertson et al. Nat. Genet. 2000 25:144-146), thegene for vitamin D24 hydrolase (Walters, M. R. Endocri. Rev. 199213:719-764), implicating this gene as the so-called “amplicon driver”for 20q13 in breast cancer. In addition, ZNF217 has been disclosed aspromoting the immortalization of mammary epithelial cells (Nonet et al.Cancer Res. 2001 61:1250-1254) and STK15 has been disclosed as atransformer (Bischoff et al. EMBO J. 1998 17:3052-3065). Aurora2 istransforming as well and is present in the 20q13 amplicon of colorectaltumors.

Another gene that maps to the 20q13 locus is EEF1A2 (Lund et al.Genomics 1996 36:359-361). EEF1A2 encodes protein elongation factorEEF1A2 (formerly eEF-1α2). During protein translation, eukaryoticelongation factors (EEF) control the recruitment of amino-acylated tRNAto the ribosome and regulate the translocation of the growingpolypeptide from the ribosome A to P sites (Hershey et al. Annu. Rev.Biochem. 1991 60:717-755). Human EEF1A2 is one of two isoforms ofeukaryotic elongation factor 1 alpha (EEF1A1 and EEF1A2) that share >90%DNA sequence and amino acid identity. EEF1A proteins bind and hydrolyzeGTP and catalyze the association of tRNAs to the ribosome during proteinelongation (Hershey et al. Annu. Rev. Biochem. 1991 60:717-755). Inaddition to their role in protein translation, EEF1A proteins from avariety of sources bind to F-actin (Condeelis, J. Trends Biochem. Sci.1995 20:169-170; Yang et al. Nature 1990 347:494-496) and depolymerizeα-tubulin microtubules (Shina et al. Science 1994 266:282-285).Accordingly, it is believed that these proteins have a role inregulating cytoskeletal organization.

A homozygous deletion of the first intron and promoter of the EEF1A2,termed the wst allele, occurs in the Wasted mouse, a spontaneous HRS/Jvariant (Shultz et al. Nature 1982 297:402-404; Chambers et al. Proc.Natl. Acad. Sci. USA 1998 95:4463-4468). The deletion prevents EEF1A2transcription (Chambers et al. Proc. Natl. Acad. Sci. USA 199895:4463-4468). EEF1A2-deficient Wasted mice suffer a B- and T-cellimmuno-deficiency and neuromuscular abnormalities (Shultz et al. Nature1982 297:402-404) and die by 30 days of age of unknown cause. Wastedmice display an increase in lymphocyte apoptosis relative to EEF1A2+/−animals and the possibility that EEF1A2 may be an inhibitor of apoptosishas been raised (Potter et al. Cell Immunol. 1998 188:111-117).

SUMMARY OF THE INVENTION

An aspect of the present invention relates to methods for diagnosing andprognosticating various cancers in a subject comprising measuring EEF1A2or EEF1A2 levels in a biological sample obtained from the subject andcomparing the measured EEF1A2 or EEF1A2 levels with levels of EEF1A2 orEEF1A2 in a control wherein an increase in the measured EEF1A2 or EEF1A2levels as compared to the control is indicative of the subject havingcancer.

Another aspect of the present invention relates to kits for detectingEEF1A2 or EEF1A2 levels in a biological sample for use in diagnosing andprognosticating cancer in a subject.

Another aspect of the present invention relates to antisenseoligonucleotides and methods of using the antisense oligonucleotides toinhibit EEF1A2 in a tumor cell.

Another aspect of the present invention relates to methods for treatingvarious cancers comprising administering to a patient suffering fromcancer an inhibitor of EEF1A2 expression and/or activity.

Yet another aspect of the present invention relates to screening assaysto identify new anticancer agents based upon the ability of an agent toinhibit EEF1A2 expression and/or activity.

DETAILED DESCRIPTION OF THE INVENTION

The genetic amplification of growth enhancing genes plays a key role inthe development of human malignancy. Important to the understanding ofoncogenesis is the identification of genes whose copy number andexpression increases during tumorigenesis. Agents that functionallyinactivate these genes or proteins encoded thereby can be used asanticancer therapeutics. Furthermore, the genes and their RNA andprotein products can be used as diagnostic and prognostic markers fordisease progression and outcome prediction.

As demonstrated herein, the present inventors have now found thatEEF1A2, the gene encoding protein elongation factor EEF1A2 (eEF-1α2), isamplified in various tumors. Further, as also demonstrated herein, thepresent inventors have also now found that EEF1A2 has properties of anoncogene in that it enhances focus formation, allows anchorageindependent growth and decreases the doubling time of fibroblasts,promotes in vivo tumorigenicity in fibroblasts and increases the growthrate and in vivo tumorigenicity of ovarian carcinoma cells whenxenografted into nude mice.

In particular, as shown herein by the inventors, EEF1A2, the geneencoding EEF1A2 (formerly eEF-1α2), is genetically amplified in 26% ofprimary ovarian and 25% of breast and colorectal tumors. In addition, asshown herein EEF1A2 amplification correlates with significantly reducedsurvival among ovarian cancer and breast cancer patients. EEF1A2 mRNAlevels are also increased in 27% of primary ovarian tumors and 33% ofestablished cell lines. The strong transforming and tumorigenicproperties of EEF1A2 are indicative of this gene and the protein encodedthereby having an important role in oncogenesis over and above anypotential role as a 20q13 amplicon driver.

Further, the inventors have now found that EEF1A2 has growth-promotingproperties. Expression of EEF1A2 alters the growth properties of mouseNIH 3T3 fibroblasts by increasing their growth rate and allowing them togrow in an anchorage-independent manner in soft agar. Expression ofEEF1A2 in RAT1 fibroblasts causes these cells to grow as a multi-layeredfocus. Anchorage-independent growth and focus formation arecharacteristics of cancerous cells. Importantly, expression of EEF1A2 inNIH 3T3 cells makes these cells tumorigenic in mice. Expression ofEEF1A2 in the human ES2 ovarian carcinoma line increases the ability ofthese human cells to grow as tumors in nude mice. Thus, it is believedthat EEF1A2 is an oncogene, a gene that promotes cancer development.

To determine whether EEF1A2 is part of the 20q13 amplicon in ovariancancer, FISH (fluorescence in situ hybridization) was used to measureEEF1A2 copy number in primary ovarian tumors. It was found that a 25%subset of primary ovarian tumors (14/53) have EEF1A2 geneamplifications. Amplifications of EEF1A2 were visualized by theincreased number of loci hybridizing to an EEF1A2 BAC (bacterialartificial chromosome) probe. The BAC probe contains the EEF1A2 3′ UTRas determined by PCR. Hybridization of a control 20p11 probe to the samesamples indicates that the increase in EEF1A2-hybridizing loci does notresult from chromosome 20 polyploidy. By chromosomal metaphase spread,it was shown that the BAC clone used for the FISH hybridizes to 20q13.Thus, EEF1A2 copy number is increased in a substantial subset of ovariantumors and is part of the 20q13 amplicon.

FISH (fluorescence-in situ hybridization) was also used to demonstratethat approximately 25% of primary human breast tumors (6 of 26) exhibitEEF1A2 gene amplification and approximately 29% of colorectal cancers (4of 14) exhibit EEF1A2 gene amplification. In contrast, EEF1A2 geneamplification was not observed in any prostate tumors (0 of 17).

To determine whether there is an increase in EEF1A2 expression inovarian tumors, northern blotting was used to measure EEF1A2 mRNA levelsin primary ovarian tumors and established ovarian carcinoma cell lines.Although the tissue-specific expression pattern of human EEF1A2 iscurrently unknown, rat and mouse EEF1A2 RNA is expressed only in normalbrain, heart and skeletal muscle (Lee et al. J. Biol. Chem. 1992267:24064-24068; Knudsen et al. Eur. J. Biochem. 1993 215:549-554).EEF1A2 message was undetectable in normal ovarian tissue, whereas 3/11primary ovarian tumors had readily detectable EEF1A2 RNA. GAPDH andEEF1A1 gene expression was similar among the samples. The EEF1A1 gene(EEF1A1) maps to 6q14, a locus not known to be involved in ovariancancer. Further, 2 out of 3 tumor samples with elevated EEF1A2 mRNA hadincreased EEF1A2 copy number. One of the tumor samples with elevatedEEF1A2 expression did not have detectable EEF1A2 amplification,suggesting that EEF1A2 expression may increase independently of EEF1A2copy number changes. EEF1A2 mRNA expression is also increased in someestablished ovarian cancer cell lines relative to normal ovarianepithelial cells. The normal ovarian epithelial cell line, NOV-61, hadundetectable EEF1A2 RNA. In contrast, 4 out of 12 ovarian tumor celllines (TOV112D, PA1, HEY, 2008) expressed EEF1A2. The OV-90, TOV81D,TOV21G, OVCAR3, OVCAR4, CAOV3, SKOV3 and ES-2 cells lines, like thenormal NOV-61 cell line, did not detectably express EEF1A2 mRNA. GAPDHand EEF1A1 gene expression was similar among the cell lines. Takentogether, these data indicate that EEF1A2 expression is increased in anapproximate 30% subset of ovarian tumor samples and cell lines.

Semi-quantitative RT-PCR (reverse-transcriptase-polymerase chainreaction) was used to estimate EEF1A2 RNA expression in primary breasttumors. EEF1A2 RNA was undetectable in normal breast tissue, but 2 outof 6 primary human breast tumors had readily detectable EEF1A2 RNAexpression. Actin expression was similar among all samples. Theincreased EEF1A2 copy number and RNA expression in primary tumorsimplicates EEF1A2 in breast cancer development.

The oncogenic properties of human EEF1A2 were also assessed. For theseexperiments, NIH 3T3 rodent fibroblast cell lines were established bystably expressing EEF1A2 under the control of the CMV promoter. TheEEF1A2 used to generate the cell lines was tagged at itscarboxy-terminus with the V5 epitope(Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr (SEQ ID NO:1))to facilitate detection by western blotting. Protein expression ofexogenous EEF1A2 was determined in three independent NIH 3T3 clones(N-1,N-2,N-3). The EEF1A2 protein in interphase cells is non-nuclear anddiffusely cytoplasmic, corresponding to the wild type localization ofthe protein (Kjaer et al. Eur. J. Biochem. 2001 268:3407-3415). TheEEF1A2-expressing clones grew as colonies in soft agar, a property notobserved in the parental NIH 3T3 cells or NIH 3T3 cells transfected withthe empty vector. Moreover, the EEF1A2-expressing clones had anaccelerated growth rate relative to the parental NIH 3T3 controls. Fourdays after plating an equal number of cells, there were approximatelyfour times as many EEF1A2-expressing cells as parental cells, indicatingthat EEF1A2 expression enhances cell growth rate.

The capacity of EEF1A2 to enhance cell growth was also assessed bymeasuring the ability of EEF1A2 to induce focus formation in Rat1fibroblasts. The ability to form foci in cell culture is a marker forcell transformation and is considered one of the general properties ofan oncogene such as RAS (Land et al. Nature 1983 Nature 304:596-602).EEF1A2 induced focus formation in Rat1 cells. The constitutively activeand transforming RAS^(va112) allele (Provencher et al. In Vitro CellDev. Biol. Anim. 2000 36:357-361) was used as a positive control. Themorphology of EEF1A2-induced foci was similar to those induced byRAS^(val12).

To determine whether EEF1A2 enhanced tumorigenicity, EEF1A2-expressingNIH 3T3 cells were subcutaneously injected into nude mice. Expression ofEEF1A2 in NIH 3T3 cells was sufficient to induce in vivo tumorigenicity.No tumor growth was observed in the parental or vector-transfected NIH3T3 cells. While the N-1 line expressed more EEF1A2 protein than eitherN-2 or N-3, it did not appear to form larger tumors in the mice nor wasit more efficient at forming colonies in soft agar. This indicates thatN-1, N-2 and N-3 are expressing enough EEF1A2 protein so that itsabundance is not the limiting factor in either anchorage-independentgrowth or in vivo tumorigenesis.

To determine the effect of EEF1A2 on an ovarian-derived cell,independent ES-2 ovarian cell lines that express EEF1A2(E-1,E-2,E-3,E-4) were generated. ES-2 are ovarian clear cell carcinomacells that do not express detectable EEF1A2 mRNA. Protein expression ofEEF1A2 was determined in four independent ES-2 derivatives. Anon-specific background band of slightly higher molecular weight thanthe EEF1A2 protein was seen in the parental and vector lanes and couldalso be discerned in the E-1, E-2, and E-3 lysates. The cell linesexpressing EEF1A2 all had an accelerated rate of tumor formation in nudemice relative to the ES-2 controls. Thus, EEF1A2 enhanced their in vivotumorigenicity. Representative sections of ES-2-derived tumors werestained with hematoxylin and eosin. All tumors showed high-grademalignancy with an ischemic necrotic core indicative of rapidtumorigenesis.

The effects of EEF1A2 on the growth and tumorigenicity of human breastcancer cells lines can also be examined. For these experiments, humanbreast cancer cell lines that over-express human EEF1A2 are developed.Since a fraction of primary breast tumors have high levels of EEF1A2expression, three breast cancer cell lines that do not expressendogenous EEF1PA2 mRNA must first be identified. To this end, northernblotting is preferably used to measure EEF1A2 RNA expression in humanbreast cancer cell lines such as MCF-7, MDA, Hs-274, Hs-280, Hs-343,Hs-362, Hs-386, Hs-739, Hs-741Hs-743, Hs-823, Hs-902, MB-157, UACC-12,HCC1008, HCC1954, BT-483, T-47D, Hs-54, HCC2157 and HCC1937 relative toexpression in the normal breast epithelial cell lines CCD-986 andCCD1056. These normal and malignant lines represent a spectrum of breasttissue types and are all available from the ATCC. However, as will beunderstood by those of skill in the art upon reading this disclosure,alternative breast cancer cell lines can be used as well. The threedifferent EEF1A2-non-expressing tumor cell lines are then transfectedwith a human EEF1A2 gene that is under the transcription control of theCMV promoter. This is the same plasmid construct used to demonstratethat EEF1A2 expression increases the tumorigenicity and in vitro growthof mouse 3T3 fibroblasts. At least three independent EEF1A2-expressingclones are derived for each cell type. Expression of the exogenousEEF1A2 after selection is determined preferably by western blotting.EEF1A2-expressing tumor cell lines are then tested for their ability togrow in soft agar. Their doubling time in 10% serum and reduced serumand plating efficiency is measured as well. To determine whether EEF1A2can directly enhance tumorigenesis EEF1A2-expressing breast cancer celllines are injected subcutaneously into nude mice and tumor volumerelative to parental non-EEF1A2-expressing cells is measured as afunction of time following injection (Ozzello, L. Prog. Clin. Biol. Res.1977 12:55-70).

The effect of EEF1A2 on the growth and tumorigenicity of normal breastepithelial cell lines can also be examined. For these experiments,normal breast epithelial cell lines expressing EEF1A2 are derived. Sincenormal epithelial cells are generally refractory to plasmidtransfection, an adenovirus EEF1A2 vector is first derived in accordancewith methods such as described by He et al. (Proc. Natl. Acad. Sci. 199895:2509-2514). The EEF1A2 adenovirus is then used to infect a breastepithelial line that does not normally express EEF1A2, as identifiedabove. EEF1A2 cell lines are assayed for in vitro growth andtumorigenicity as described above for the malignant cells. Since normalbreast epithelial cells generally have a finite life-span, the doublingpotential of an EEF1A2 expressing cell line can also be comparedrelative to that of a cell line infected with a control lacz adenovirus.

The demonstrated ability herein of EEF1A2 promoting cancerous growth isindicative of EEF1A2 being a target for anti-cancer therapy. It isbelieved that EEF1A2 inactivation through inhibition of expression ofEEF1A2 and/or through inhibition of the activity of this protein willslow or stop the growth of cancer cells. Accordingly, one aspect of thepresent invention relates to methods of treating cancer by administeringan agent that inhibits EEF1A2 expression and/or activity. Such EEF1A2inactivating agents are expected to be particularly useful in thetreatment of ovarian cancer. Other cancers, including breast andcolorectal cancer, are also expected to be targets for EEF1A2inactivation.

As used herein, “expression of EEF1A2”, i.e., gene expression, refers toeither or both mRNA products of the EEF1A2 gene and consequent proteinproducts. Gene expression may be measured therefore by measuring mRNAand/or protein levels. Alternatively, expression may be monitored byassaying protein activity.

In one embodiment of a method for treatment, an anticancer agentcomprises an antisense oligonucleotide, which hybridizes to EEF1A2 ormRNA thereof and inhibits transcription of EEF1A2 and/or proteintranslation of EEF1A2 mRNA. An antisense oligonucleotide can comprise anucleotide sequence which is complementary to a coding strand of anucleic acid, e.g. complementary to an mRNA sequence, constructedaccording to the rules of Watson and Crick base pairing, and canhydrogen bond to the coding strand of the nucleic acid. The antisensesequence complementary to a sequence of an mRNA can be complementary toa sequence found in the coding region of the mRNA or can becomplementary to a 5′ or 3′ untranslated region of the mRNA.Furthermore, an antisense oligonucleotide can be complementary insequence to a regulatory region of the gene encoding the mRNA, forinstance a transcription initiation sequence or regulatory element.Preferably, an antisense nucleic acid complementary to a regionpreceding or spanning the initiation codon or in the 3′ untranslatedregion of an mRNA is used. An antisense nucleic acid can be designedbased upon the nucleotide sequence shown in SEQ ID NO: 5. A nucleic acidis designed which has a sequence complementary to a sequence of thecoding or untranslated region of the shown nucleic acid.

The antisense oligonucleotides of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. The antisense oligonucleotide can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids e.g. phosphorothioatederivatives, acridine substituted nucleotides, and 2′-O-propyl modifiednucleotides can be used. Alternatively, the antisense oligonucleotidescan be produced biologically using an expression vector into which anucleic acid has been subcloned in an antisense orientation (i.e.nucleic acid transcribed from the inserted nucleic acid will be of anantisense orientation to a target nucleic acid of interest). Theantisense expression vector is introduced into cells in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

In some embodiments of the present invention, the antisenseoligonucleotide may be formulated in liposomes and delivered to targetcells. Methods of manufacturing liposomes are described, e.g., in U.S.Pat. No. 4,522,811, No. 5,374,548 and No. 5,399,331. The liposomes maycomprise one or more targeting moieties that are selectively transportedinto specific cells or organs (see, e.g., Ranade, J. Clin. Pharmacol.1989 29: 685). Exemplary targeting moieties include folate, biotin,mannosides, antibodies, surfactant protein A receptor, and gp120. Theantisense oligonucleotides may be subjected to electrostatic liposomeencapsulation (NeoPharm Inc., Lake Forest, Ill.; Gokhale et al. ClinicalCancer Research 2002 8:3611-3621). Such liposomes may comprise atargeting moiety, e.g., a tumor-specific monoclonal antibody.

The antisense oligonucleotides of the present invention are useful forinhibiting expression of nucleic acids (e.g. mRNAs) encoding proteinshaving EEF1A2 activity, thereby decreasing expression of proteins havingEEF1A2 activity. Since increased expression of proteins having EEF1A2activity is associated with and can confer oncogenic properties on acell including enhancing focus formation, allowing anchorage independentgrowth and decreasing the doubling time of fibroblasts, promoting invivo tumorigenicity in fibroblasts and increasing the growth rate and invivo tumorigenicity of carcinoma cells xenografted into nude mice,decreasing expression of such proteins can inhibit or reverse suchoncogenic properties of the cell into which the antisenseoligonucleotide has been introduced. Antisense oligonucleotides can beintroduced in to a cancer cell, preferably an ovarian, breast orcolorectal cancer cell in culture to inhibit EEF1A2 expression. One ormore antisense nucleic acids, such as oligonucleotides, can be added tocells in culture media, typically at 10 to 1000 μg/ml. A cultured cancercell in which EEF1A2 expression is inhibited is useful for testing theefficacy of potential therapeutic agents. For example, EEF1A2 expressioncould be inhibited in a tumor cell line that expresses EEF1A2 todetermine the contribution of EEF1A2 to an observed response of the cellto a particular therapeutic agent.

The antisense oligonucleotides of the present invention can also be usedin gene therapy to correct or prevent EEF1A2 expression in a subject.For example, antisense sequences can be used to render malignant cellsincapable of expressing EEF1A2. Correction or prevention of EEF1A2expression is expected to be useful in the treatment of cancers, and inparticular ovarian, breast and colorectal cancers. Administration ofantisense nucleic acids to a subject may be most effective when theantisense oligonucleotide is contained in a recombinant expressionvector which allows for continuous production of antisense RNA.Recombinant molecules comprising an antisense oligonucleotide, can bedirectly introduced into tissues, including ovarian, breast orcolorectal tissue in vivo, using delivery vehicles such as liposomes,retroviral vectors, adenoviral vectors and DNA virus vectors. A deliveryvehicle can be chosen which can be targeted to a cell of interest in thesubject (e.g. a ovarian, breast or colorectal tumor cell). Antisenseoligonucleotides can also be introduced into isolated cells, such asthose of the hematopoietic system, ex vivo using viral vectors ortechniques such as microinjection, electroporation, coprecipitation andincorporation of DNA into liposomes (e.g. lipofectin) and such cells canbe returned to the donor. Recombinant molecules can also be delivered inthe form of an aerosol or by lavage. Antisense oligonucleotides can alsobe delivered by other routes including, but not limited tointravenously, orally, topically, rectally, intramuscularly andintraperitoneally, in accordance with well known procedures.

Antisense inactivation of EEF1A2 was achieved using two EEF1A2-specificphosphorothioated antisense oligonucleotides. Phosphorothioated DNAoligonucleotides were used as these modified oligonucleotides arenon-toxic in humans, nuclease resistant, stable in vivo and in vitro,are readily taken up by cells and activate rapid degradation of the mRNAtarget (Gewirtz et al. Blood 1998 92:712-736; Agrawal et al. Proc. Natl.Acad. Sci. USA 1990 87:1401-1405). However, as will be understood bythose of skill in the art upon reading this disclosure, othermodifications well known to those skilled in the art can be used.

These antisense oligonucleotides comprised the following sequences:CTTTTGCTGGGAGTGTGAGG; (SEQ ID NO:2) and GCTGGGAGTGTGTGAGGGGCTG. (SEQ IDNO:3)Both antisense oligonucleotides decreased EEF1A2 mRNA levels in humanRamos cells. mRNA levels of EEF1A1 were not decreased by administrationof these antisense oligonucleotides. A control sequence,GGTTGCTGTGGGCTTGAGT (SEQ ID NO:4) had no effect on mRNA levels of EEF1A2or EEF1A1 in the human Ramos cells.

Accordingly, the invention provides a method for inhibiting EEF1A2 of atumor cell, preferably an ovarian, breast or colorectal tumor cell byintroducing into the tumor cell a nucleic acid which is antisense to anucleic acid which encodes the protein shown in SEQ ID NO: 6. In apreferred embodiment, the antisense oligonucleotide comprises SEQ IDNO:2 or SEQ ID NO:3. Further, the method may comprise administration ofa second therapeutic drug, preferably a second anticancer drug such as ataxane, which as taught infra, is expected to have reduced efficacy intumor cells expressing EEF1A2. Accordingly, administration of anantisense oligonucleotide that inhibits EEF1A2 in combination with ananticancer drug, preferably a taxane, is expected to enhance efficacy ofthe anticancer drug.

Additional antisense oligonucleotides to those exemplified herein, alsocapable of hybridizing to EEF1A2 or mRNA thereof and inhibitingtranscription of EEF1A2 and/or protein translation of EEF1A2 mRNA can beidentified routinely by those skilled in the art in accordance with theteachings herein.

The specific inactivation of EEF1A2 requires an antisense molecule thatbinds to EEF1A2 (Genbank Accession No. NM_(—)001958; SEQ ID NO:5) butnot the closely related EEF1A1 (Genbank Accession No. NM_(—)001402; SEQID NO:7). The mRNA coding sequence of EEF1A1 and EEF1A2 are >90%identical. The 5′ and 3′ ends of the human EEF1A2 mRNA are the mostdifferent from EEF1A1 mRNA, making the mRNA termini preferred targetsfor specific inactivation of EEF1A2 and design of additional antisenseoligonucleotide. To identify additional useful EEF1A2 specific antisenseoligonucleotide, approximately 15 EEF1A2 specific antisense moleculesare prepared; 9 complementary to the 5′ end (residues 1-39 and 52-78)and 6 complementary to the 3′ end (residues 1467-1500).

Northern blotting is used to measure the ability of each antisense, indoses of 1-20 μM, to decrease EEF1A2 but not EEF1A1 mRNA. GenePorter(Gene Therapy Systems) is preferably used to deliver the antisenseoligonucleotide since its delivery efficiency is >80% with minimalcytotoxicity. Transfection reagents are not required for in vivodelivery. Three ovarian or breast cancer cell lines that have highEEF1A2 expression are used as well as one non-EEF1A2-expressing cellline to determine the ability of each antisense to reduce EEF1A2 but notEEF1A1 mRNA, to halt cell cycle (propidium iodide staining), induceapoptosis (Annexin V staining; vanEngeland et al. Cytometry 1998 31:1-9)and decrease overall viability (trypan blue exclusion) of the cancercell lines.

Because germline EEF1A2 inactivation causes immune dysfunction andmortality in Wasted mice (Shultz et al. Nature 1982 297:402-404), theantisense oligonucleotides are tested for systemic toxicity in nude andnormal C57BL/6 mice. The goal is to determine the maximum tolerated doseof each antisense compound. To this end, mice (n=4/dose) are treatedwith decreasing doses, in saline, from 6.0 mg/kg i.v. every day for 2weeks. This dose regimen represents the maximum tested dose forinhibition of tumorigenesis by a c-raf antisense in a mouse model ofA549 lung carcinoma model (Monia et al. Nat. Med. 1996 2:668-675) and isused as an experimental starting point. Dose can be increased if theantisense is well tolerated. During treatment the mice are observeddaily for malaise, mobility difficulties or other treatment sideeffects. The amount of circulating antibodies and peripheral B- andT-cells are monitored weekly using ELISA assays. Animals are alsoweighed daily. At the conclusion of treatment, organs are weighed.Antisense oligonucleotides that do not show evidence for toxicity arethen further studied.

For example, the anti-breast or anti-ovarian tumor activity of theantisense oligonucleotide is examined in mice injected subcutaneouslywith two independent EEF1A2-expressing human breast or ovarian cancercells. After a measurable tumor volume (>5 mm in diameter) has appeared,the antisense molecule is delivered intravenously to the reconstitutedmice at the highest non-toxic dose as determined above (n=6 mice/dose).Tumor volume is monitored daily. As appropriate, a minimum effectivedose is determined. The ability of the antisense oligonucleotide to haltor reverse tumor progression is indicative of its efficacy.

The pharmacokinetic properties of the antisense in mice can also beexamined using ³²P-labeled antisense delivered iv. Plasma half-life andurine and fecal clearance rates can be determined as well asaccumulation rates in the brain, thymus, spleen, bone, liver, andkidney.

The nucleic acids of the invention can further be used to designribozymes which are capable of cleaving a single-stranded nucleic acidencoding a protein having EEF1A2 activity, such as an mRNA. A catalyticRNA (ribozyme) having ribonuclease activity can be designed which hasspecificity for a EEF1A2-encoding mRNA based upon the sequence of anucleic acid of the invention. For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the base sequenceof the active site is complementary to the base sequence to be cleavedin a EEF1A2-encoding mRNA. See for example Cech et al. U.S. Pat. No.4,987,071: Cech et al. U.S. Pat. No. 5,116,742. Alternatively, a nucleicacid of the invention could be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See forexample Bartel, D. and Szostak, J. W. Science 261, 1411-1418 (1993).

Short interfering RNA (siRNA) can be also be used to inhibit EEF1A2.siRNA is a relatively new technology that silences gene expressionthrough a process referred to as RNA interference (RNAi) (Moss, E. G.Curr. Biol. 2001 11:R772-R775; Carthew, R. W. Curr. Opin. Cell Biol.2001 13:244-248). RNAi depends on the formation of double stranded RNA(dsRNA) derived from coding sequences of the gene to be silenced (Moss,E. G. Curr. Biol. 2001 11:R772-R775; Carthew, R. W. Curr. Opin. CellBiol. 2001 13:244-248). Gene expression is halted through a combinationof target gene methylation (Wassenegger et al. Cell 1994 76:567-576) andpost-transcriptional degradation of the gene's mRNA (Hammond et al.Nature 2000 404:293-296). RNAi can be experimentally activated throughthe use of siRNA, duplexes of 21-25 base pair RNA fragments that arecomplementary to the gene being silenced (Elbashir et al. Nature 2001411:494-498). siRNA can be used to inactivate gene expression incultured mammalian cell lines (Caplen et al. Proc. Natl. Acad. Sci. USA2001 98:9742-9747; Cogoni, C. and Macino, G. Curr. Opin. Genet. Dev.2000 10:638-643) and in multiple organs of postnatal mice (Lewis et al.Nat. Genet. 2002 32:107-108). siRNA-mediated gene-silencing effect isusually achieved using double-stranded siRNA 21 nucleotides in length.siRNA usually consist of a 19-nucleotide complementary region and a twonucleotide non-complementary 3′ overhang. An effective RNAi targetsequence is likely to be 50-100 nucleotides downstream of the startcodon. siRNA are commercially synthesized (Xeragon) and then transfectedinto EEF1A2-expressing breast or ovarian tumors cells usingTransMessenger (Qiagen), a lipid based transfection agent designedspecifically for delivery of RNA. Northern and western blots are used todetermine EEF1A1 and EEF1A2 mRNA and protein levels. A plasmid basedvector system is used to generate siRNA since a plasmid vector isgenerally deliverable to animals either on its own or incorporated intoa virus. Like the antisense experiments described above, the ability ofeach siRNA to reduce EEF1A2 mRNA, to halt cell cycle, induce apoptosisand decrease overall viability of the breast or ovarian cancer celllines is determined.

Other anticancer agents useful in the present invention may comprisesmall organic molecules, proteins, peptides or peptidomimetics that arecapable of inactivating or inhibiting EEF1A2. High-throughput technologyis used to screen libraries of several thousand compounds, e.g. naturalproduct libraries, to identify inactivators or inhibitors of EEF1A2. Thescreen assays the effect that each compound of the library has on thegrowth rate of cultured cell lines, preferably EEF1A2-overexpressingcells. Screening with high-throughput technology is currently ongoingand EEF1A2 inhibition has been detected in a library of compounds. Likethe antisense experiments described above, the ability of each smallmolecule to halt cell cycle, induce apoptosis and decrease overallviability of the breast or ovarian cancer cell lines is then determined.In addition, the ability of the molecule to inhibit enhancement of thetranslation of polyPhe mRNA by purified EEF1A2 in rabbit reticulocytelysates, to bind and hydrolyze radiolabelled GTP or to co-precipitatewith F-actin, is assessed. Such in vitro assays are allwell-characterized for assessing EEF1A function.

The present invention also relates to screening assays for use inidentifying potential anticancer agents based upon their ability toinactivate or inhibit EEF1A2. For example, a screening assay of thepresent invention may comprise individually testing potential anticanceragents for their ability to inhibit: a) EEF1A2-mediated enhancement ofNIH 3T3 cell growth; b) EEF1A2-mediated enhancement of proteintranslation; and/or c) EEF1A2-mediated microtubule cleavage. The abilityof a test agent to inhibit one or more of these activities is indicativeof the agent being useful in the treatment of cancer, particularlyovarian, breast or colorectal cancer.

Another aspect of the present invention relates to the use of EEF1A2gene amplification, EEF1A2 mRNA levels or EEF1A2 protein levels oractivity as a prognostic marker in cancer, particularly ovarian cancer,as well as breast and colorectal. Ovarian cancer patients with EEF1A2amplification survived a shorter period of time following diagnosis thanovarian cancer patients without EE1FA2 amplification. EEF1A2 geneamplification is also associated with decreased probability of 10-yearsurvival in breast cancer patients. Thus, detection of EE1FA2amplification or increases in EE1FA2 mRNA levels or EEF1A2 proteinslevels or activity can be used to prognosticate survival time of acancer patient.

FISH and immunohistochemistry can be used as well to determine theextent to which EEF1A2 gene amplification and protein expressioncorrelate with 11-year relapse-free survival, 11-year survival andbreast tumor size, grade, nodal status, grade, estrogen receptor (ER)status, Her2 status, and lymphovascular invasion in a cohort of >1500Canadian breast tumor samples (Parker et al. Am. J. Clin. Pathol. 2002117:723-728). More specifically, EEF1A2 can be linked to the abovelisted clinical and pathological parameters using a tissue microarray(TMA) of >5000 Canadian breast tumor samples. TMA are composed of 600 μmdiameter cylindrical samples taken from different archival tissue blocksand placed into a single empty recipient paraffin block (Bubendorf etal. J. Pathol. 2001 195:72-79; Kononen et al. Nat. Med. 1998 4:844-847).A TMA typically contains several hundred different tumor samples thatcan be simultaneously analyzed by immunohistochemistry or in situhybridization in a single TMA section on a single standard microscopeslide. TMA technology has proved effective at analyzing the molecularpathology of breast, bladder, and prostate cancer and substantiallyreduces the time required to establish correlations between tumorpathology and molecular biology. For this analysis, a TMA of >5000Canadian breast tumor samples will be used which have 11-yearrelapse-free survival, 11-year survival and breast tumor size, grade,nodal status, grade, estrogen receptor (ER) status, Her2 status, andlymphovascular invasion data associated with them (Parker et al. Am. J.Clin. Pathol. 2002 117:723-728).

In one embodiment, the presence of EEF1A2 amplifications in primaryovarian and breast tumors is identified using an EEF1A2-containingbacterial artificial chromosome (BAC). The presence of EEF1A2amplifications can be used as a genetic marker to predict theprobability of survival.

In another embodiment EEF1A2 protein expression can serve as theprognostic marker of ovarian, breast or colorectal cancer. For example,the EEF1A2 protein is not expressed in normal ovarian epithelial cellsor breast tissue. Thus, antibodies that specifically recognize EEF1A2protein can be generated and used to stain samples of tumor removed fromovarian cancer or breast cancer patients.

In one embodiment, to produce these polyclonal antibodies, rabbits areimmunized with the SHTTLLEAVDCIL (SEQ ID NO:8) peptide conjugated to KLH(keyhole limpet hemocyanin). This peptide, which corresponds to EEF1A2residues 224-236, contains 4 amino acid differences between EEF1A1 andEEF1A2, and is found in a predicted hinge region between the actinbinding and tRNA binding domains. EEF1A1 specific antibodies from theimmunization are absorbed using an Affigel affinity column containingSGVSLLEALDTIL (SEQ ID NO:9; differences underlined), the EEF1A1 peptide.Specificity of the antibody is confirmed by immunoblotting with aGST-EEF1A1 and a GST-EEF1A2.

Patients with tumor samples that are positive for EEF1A2 are expected tosurvive for a shorter period of time as compared to patients with tumorsample negative for EEF1A2. Prognostic information relating toEEF1A2-gene amplification and/or EEF1A2 protein expression can be usedto enhance clinical decision-making and to select appropriate treatmentregimes. NIH 3T3 cells ectopically expressing EEF1A2 are resistant tothe apoptosis-induced by cisplatin and staurosporine. Further, micelacking EEF1A2 show increased lymphoid apoptosis. Accordingly, it isbelieved that EEF1A2 modulates the sensitivity of cancer cells toselected treatments.

Furthermore, since EEF1A can directly or indirectly cause microtubulesevering (Shiina et al. Science 1994 266:282-285), it is believed thatEEF1A2 modulates the cytotoxicity of taxane compounds since theircytotoxicity stems from their ability to stabilize microtubules. Inparticular, EEF1A2 expression is expected to increase resistance totaxol-induced cell death and microtubule stabilization and thus isprognostic of a poor response to taxol. Examples of other anticancertreatments, efficacy of which may be modulated by the EEF1A2 expressioninclude, but are not limited to, classes of compounds such asanthracyclines, epipodophyllotoxins, vinca alkaloids, metallocenes (suchas platinum-based compounds), of which cyclophosphamide, methotrexate,fluorouracil, doxorubicin, epirubicin, paclitaxel and cisplatin areexamples.

In addition, increased EEF1A2 expression in tumors such as primaryovarian tumors in a subject is expected to lead to increased EEF1A2protein levels in biological samples such as blood and other tissuesobtained from the subject. Accordingly, measurement of increased EEF1A2levels in a biological sample such as plasma, serum or other tissueobtained from a subject can be used as a diagnostic tool for cancerssuch as ovarian, breast and colorectal cancer in the subject. Blood orother tissue samples can be taken from a subject and analyzed for thepresence of EEF1A2 protein using a standard immunoassay technique suchas an ELISA with an EEF1A2-specific antibody. Measured levels of EEF1A2protein in the sample can then be compared to levels in a control. Asused herein, by “control” it is meant, a sample obtained from anindividual known not have cancer, a sample obtained previously from thesubject prior to the onset or suspicion of cancer, or a standard fromdata obtained from a data bank corresponding to currently acceptednormal levels of this gene or gene product. Increased EEF1A2 proteinlevels in the sample obtained from the subject as compared to levels inthe control are indicative of the subject having ovarian, breast orcolorectal cancer. The comparison performed may be a straight-forwardcomparison, such as a ratio, or it may involve weighting of one or moreof the measures relative to, for example, their importance to theparticular situation under consideration. The comparison may alsoinvolve subjecting the measurement data to any appropriate statisticalanalysis.

Another aspect of the present invention relates to kits for diagnosingand prognosticating cancer in a subject by detecting EEF1A2 geneamplification or EEF1A2 expression and/or activity. Kits for detectionof EEF1A2 gene amplification preferably comprise a means for detectionsuch as a EEF1A2-containing bacterial artificial chromosome (BAC) aswell as instructions for use of BAC in detecting EEF1A2 geneamplification in tumor tissue samples. Kits for detection of EEF1A2 mRNAlevels preferably comprise a means for detection such as, for example,northern blotting or a gene chip, as well as instruction for use ofnorthern blotting or the gene chip in detecting EEF1A2 mRNA levels. Kitsfor detection of EEF1A2 protein levels preferably comprise a means fordetection such as an antibody specific for EEF1A2 as well asinstructions for use of such antibody to immunoassay a biological samplesuch as a tumor tissue biopsy sample, or a serum or blood sampleobtained from a subject for the presence of EEF1A2. Other componentsincluded in these kits may comprise EEF1A2 standards, dilutingsolutions, and/or wash buffers routinely provided in diagnostic andprognostic kits of this nature.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Fluorescence Hybridization and Microscopy

Fluorescence in situ hybridization (FISH) was performed in accordancewith the procedure described by Demetrick, D. J. (Mod. Pathol. 19969:133-136). In these experiments, EEF1A2 and 20p11 BAC clones werelabeled with FITC-dUTP and Digoxygenin (DIG)-dUTP, respectively. Thelabeled clones were then hybridized at 37° C. to interphase nuclei fromfrozen ovarian carcinoma tissue samples. Slides were counter-stainedwith DAPI and visualized utilizing a Zeiss Axioplan 2 microscope. APhotometrics PXL 1400 CCD camera was used to capture images ofrepresentative interphase nuclei and Electronic Photography version 1.3Biological Detection software used for alignment. Adobe PhotoShop wasused to pseudocolor FITC and DIG labeled probes. A V5 antibody(InVitrogen) diluted 1:500 in phosphate buffered saline (PBS) followedby an Alexa 546-conjugated (1:200 in PBS) secondary antibody was used todetermine EEF1A2 localization.

Example 2 RNA Purification and Northern Blotting

Ovarian tumor samples were obtained from the Gynecology and OncologyGroup of the Cooperative Human Tissue Network. RNA was prepared from100-200 mg of frozen tumor homogenized in 2 ml of TriZol (Gibco) as perthe manufacturer's directions. RNA from cell lines was obtained throughlysis of a 60 mm plate with 1 ml of Trizol (Gibco). 10 ug of total RNAwas loaded per lane and RNA was transferred to GeneScreen. Normal ovarymRNA was obtained from Stratagene. Membranes were pre-hybridized at 63°C. in 25 ml Church's Buffer, hybridized in 15 ml Church's at 59° C.overnight, and washed at 62° C. The EEF1A2 probe was a 598 BamHI/PstIfragment of the human EEF1A2 cDNA.

Example 3 Cell Culture and Western Blotting

NIH 3T3 and ES-2 cells were grown in 10% FBS/DMEM and 10% FBS/McCoy's 5Arespectively. EEF1A2-expressing NIH 3T3 and ES-2 cells were derived bytransfecting NIH 3T3 cells with 5 ug of the EEF1A2 plasmid and 15 ul ofSuperFect (Qiagen) per 60 mm dish. 0.4 mg/ml Zeocin (InVitrogen) wasused to select transfectants and independent clones derived by limitingdilution cloning. An α-V5 antibody (InVitrogen; 1:500 in TBST) followedby an HRP conjugated goat anti-mouse (BioRad; 1:1,000 in TBST) and ECL+(Amersham) were used to detect EEF1A2 expression. Cell growth wasmeasured by Coulter counting triplicate independent platings from a NUNC6-well plate. For focus-forming assays, EEF1A2 and RAS^(val12), bothunder the control of the CMV promoter, were transfected into Rat1fibroblasts using calcium phosphate according to the manufacturer'sdirections (Gibco). The pCDNA3 empty vector was used as a control.Transfected cells were grown in 2% FBS/DMBM at 37° C. for 14 days andthe media changed every three days. Transfection efficiency wasdetermined by counting colonies that arise in selective media (Zeocinfor EEF1A2 and G418 for Ras). Foci were counted by washing plates inPBS, fixing in 10% acetic acid and staining with 0.4% crystal violet.Counts are the mean of triplicate experiments, each containingtriplicate independent transfections. For soft agarose assays, 2×10⁴ NIH3T3 cells were placed in 3 ml of 0.35% low gelling temperature agarose(Sigma) in 10% FBS/DMEM and overlaid on 5 ml 0.8% agarose/10% FBS/DMEMin a 60 mm dish. Cells were grown at 37° C. for 14 days to allow colonyformation.

Example 4 Tumor Xenografts

NIH 3T3 or ES-2 cells (1×10⁶) were injected subcutaneously into the hindleg of nude mice and the animals were sacrificed 21 days post injection.Tumor volume (V) was estimated from the length (l) and width (w) of thetumor by the formula: V=(π/6)×((l+w)/2)³. Tumors were fixed in formalinovernight at 4° C. and paraffin embedded. Sections were de-waxed andstained with Haematoxylin and Eosin. Animal experiments were conductedthrough protocols approved by the Central Animal Facility at McMasterUniversity.

1. A method for diagnosing cancer in a subject comprising measuringEEF1A2 gene amplification, EEF1A2 mRNA levels, or EEF1A2 protein levelsor activity in a biological sample obtained from the subject andcomparing the measured EEF1A2 gene amplification, EEF1A2 mRNA levels, orEEF1A2 protein levels or activity with levels of EEF1A2 geneamplification, EEF1A2 mRNA levels, or EEF1A2 protein levels or activityin a control wherein an increase in the measured EEF1A2 geneamplification, EEF1A2 mRNA levels, or EEF1A2 protein levels or activityas compared to the control is indicative of the subject having cancer.2. The method of claim 1 wherein the cancer is ovarian cancer, breastcancer or colorectal cancer.
 3. The method of claim 1 wherein thebiological sample comprises a serum or plasma sample or a tumor tissuebiopsy sample obtained from the subject.
 4. A method for prognosticatingsurvival and selecting an effective treatment regime for a patientsuffering from cancer comprising measuring EEF1A2 gene amplification,EEF1A2 mRNA levels, or EEF1A2 protein levels or activity in a biologicalsample obtained from the subject.
 5. The method of claim 4 wherein thepatient is suffering from ovarian, breast or colorectal cancer.
 6. Themethod of claim 4 wherein the biological sample comprises a serum orplasma sample or a tumor tissue biopsy sample obtained from the patient.7. A kit for prognosticating and/or diagnosing cancer comprising meansfor measuring EEF1A2 gene amplification, EEF1A2 mRNA levels, or EEF1A2protein levels or activity in a biological sample.
 8. A method forinhibiting expression of EFF1A2 of a tumor cell, comprising contactingthe cell with an antisense oligonucleotide which inhibits expression ofEEF1A2 by the cell, wherein a protein encoded by said EEF1A2 has anamino acid sequence comprising SEQ ID NO:6, and wherein the antisenseoligonucleotide specifically inhibits expression of said EEF1A2 and doesnot inhibit expression of EEF1A1.
 9. The method of claim 8, wherein theantisense oligonucleotide interacts in the cell with an mRNA moleculewhich encodes said EEF1A2 protein such that expression of the protein inthe cell is inhibited.
 10. The method of claim 8, wherein the antisenseoligonucleotide is a ribozyme.
 11. The method of claim 8, wherein thetumor cell is an ovarian, breast or colorectal tumor cell.
 12. Themethod of claim 8, further comprising exposing the tumor cell to atherapeutic drug such that growth of the tumor cell is inhibited. 13.The method of claim 12, wherein the therapeutic drug comprises ananthracycline, epipodophyllotoxin, vinca alkaloid, or metallocene, or iscyclophosphamide, methotrexate, fluorouracil, doxorubicin, epirubicin,paclitaxel, cisplatin, or staurosporine.
 14. The method of claim 12,wherein the therapeutic drug comprises a taxane.
 15. The method of claim8, wherein the antisense oligonucleotide is included in a recombinantexpression vector which permits expression of the antisenseoligonucleotide in the cell.
 16. The method of claim 8, wherein theantisense oligonucleotide is administered in a viral vector or liposome.17. The method of claim 8, wherein the antisense oligonucleotide bindsto a coding region of a nucleic acid molecule which encodes said EEF1A2protein.
 18. The method of claim 8, wherein the antisense nucleicoligonucleotide binds to a non-coding region of a nucleic acid moleculewhich encodes said EEF1A2 protein.
 19. The method of claim 8, whereinthe antisense oligonucleotide is complementary in sequence to aregulatory region of a gene which encodes said EEF1A2 protein.
 20. Themethod of claim 8, wherein the antisense oligonucleotide iscomplementary in sequence to a transcription initiation region of a genewhich encodes said EEF1A2 protein.
 21. The method of claim 8, whereinthe antisense oligonucleotide is complementary in sequence to a regionwhich precedes or spans the translation initiation codon of a gene whichencodes said EEF1A2 protein.
 22. The method of claim 8, wherein theantisense oligonucleotide is complementary in sequence to anuntranslated region of a mRNA which encodes said EEF1A2 protein.
 23. Themethod of claim 22, wherein the antisense oligonucleotide iscomplementary in sequence to a 3′ untranslated region of the mRNA.
 24. Amethod for treating cancer comprising administering to a patientsuffering from cancer an inhibitor of EEF1A2 expression and/or EEF1A2activity.
 25. The method of claim 24, wherein the inhibitor of EEF1A2expression or EEF1A2 activity comprises an antisense oligonucleotidewhich inhibits expression of an EEF1A2 protein by a tumor cell.
 26. Themethod of claim 24 or 25, further comprising administering a therapeuticdrug.
 27. The method of claim 26, wherein the therapeutic drug comprisesan anthracycline, epipodophyllotoxin, vinca alkaloid, or metallocene, oris cyclophosphamide, methotrexate, fluorouracil, doxorubicin,epirubicin, paclitaxel, cisplatin, or staurosporine.
 28. The method ofclaim 26, wherein the therapeutic drug comprises a taxane.
 29. Ascreening assay to identify new anticancer agents comprising measuringan agent's ability to inhibit EEF1A2 expression and/or EEF1A2 activity.30. The screening assay of claim 29 wherein the agent's ability toinhibit EEF1A2 expression and/or EEF1A2 activity is measured byEEF1A2-mediated enhancement of NIH 3T3 cell growth, EEF1A2-mediatedenhancement of protein translation or EEF1A2-mediated microtubulecleavage.